Fig. 5: Predicted domain architecture, folding and tyrosine phosphorylation sites of MACC1.

A Overview of structural domains (colored boxes) and interaction motifs (white boxes) predicted by sequence alignment. ZU5 (green), SH3 (yellow), variant Src homology 3 domain; DD (red), death domain; NPF, Epsin homology 15 interacting motif; DPF, adaptor protein 2α interacting motif; KxxPxxP, proline-rich motif, class I SH3 interaction motif. B, C Predicted folding of MACC1, according to sequence alignment, comparison with known structures and modeling of highly similar domains. The N-terminus (aa 1–199) of MACC1 was predicted to be highly disordered and was omitted in the representation. Backbone view (B) and space filling representation with electrostatic charge distribution (C) are displayed from three different angles. Backbone representation (B): ß-sandwich structure overlapping with the predicted SH3 domain (yellow); 2 adjacent similar folds overlapping the predicted ZU5 domain (green); coiled coil structure overlapping with both predicted death domains (red). Space filling representation (C): negatively charged/acidic areas (red); positively charged/basic patches (blue); neutral/hydrophobic areas (white). D Position of three tyrosine residues (Y673, Y695, Y793) within the coiled coil structure, which are predicted sites for tyrosine phosphorylation. All three tyrosine residues are predicted to face the solvent and are accessible for modifying enzymes. Backbone representation of the coiled coil structure (red); tyrosine residues in licorice representation (green). E, F Quantification of the MACC1 tyrosine phosphorylation pattern was performed by SRM-based MS. Phosphorylation of tyrosine residue Y793 (E) and Y695 (F) was determined in SW480/vector, SW480/MACC1-wt, SW480/Y673AF, SW480/Y693F, SW480/Y793F, and SW480/Y3xF cells. SDM of the sites Y793 and Y695 of MACC1 restricted phosphorylation. Phosphorylation of position Y793 was independent of phosphorylation at Y673 and Y695, whereas phosphorylation of Y695 was dependent on phosphorylation at positions Y673 and Y793 demonstrating a hierarchy of phosphorylation events. G Ectopic overexpression of constitutively active MEK1 increases phosphorylation of MACC1 (peptide VSpYVIK) in HEK293T cells. MS was performed three independent times, with three technical replicates in each experiment. Given are the relative amounts of MACC1 peptide VSpYVIK compared to control cells, expressed as mean with SD. Western Blot analyses confirm MEK1 S218D S222D overexpression. H To analyze the impact of HGF treatment on phosphorylation of endogenous MACC1, SW620 cells with endogenous MEK1 expression and high endogenous MACC1 level were treated with HGF. Cells were serum starved for 4 h and subjected to 20 or 100 U of HGF. After indicated incubation times cells were harvested and lysed in RIPA buffer supplemented with phosphatase and protease inhibitors. MACC1 was isolated using IP. Amounts of phosphorylated MACC1 (peptide VSpYVIK) were quantified with SRM-MS. All treatments led to significantly increases in pY MACC1. Values are shown relative to control cells without HGF treatment with SD. Significance was analyzed using ANOVA with the Dunnett’s multiple comparison test.